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. Author manuscript; available in PMC: 2022 Jan 2.
Published in final edited form as: Virology. 2020 Oct 6;552:73–82. doi: 10.1016/j.virol.2020.09.001

Generation and Preliminary Characterization of Vertebrate-Specific Replication-Defective Zika Virus

Shengfeng Wan a,b,e,†, Shengbo Cao a,†, Xugang Wang a, Yanfei Zhou c, Weidong Yan a,b, Xinbin Gu b, Tzyy-Choou Wu d, Xiaowu Pang b,*
PMCID: PMC7733535  NIHMSID: NIHMS1642935  PMID: 33075709

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that replicates in both vertebrate and insect cells, whereas insect-specific flaviviruses (ISF) replicate only in insect cells. We sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus for the eventual development of a safe ZIKV vaccine. Reverse genetics was used to introduce specific mutations into the furin cleavage motif within the ZIKV pre-membrane protein (prM). Mutant clones were selected, which replicated well in C6/36 insect cells but exhibited reduced replication in non-human primate (Vero) cells. Further characterization of the furin cleavage site mutants indicated they replicated poorly in both human (HeLa, U251), and baby hamster kidney (BHK-21) cells. One clone with the induced mutation in the prM protein and at positions 291and 452 within the NS3 protein was totally and stably replication-defective in vertebrate cells (VSRD-ZIKV). Preliminary studies in ZIKV sensitive, immunodeficient mice demonstrated that VSRD-ZIKV-infected mice survived and were virus-negative. Our study indicates that a reverse genetic approach targeting the furin cleavage site in prM can be used to select an insect-specific ZIKV with the potential utility as a vaccine strain.

Keywords: Zika virus, Vertebrate-specific replication-defective, Furin, prM cleavage, Viral tropism, Vaccine

1. Introduction

Zika virus (ZIKV) is an arthropod-borne flavivirus that emerged as a global threat to humans (Fauci and Morens, 2016). The development of a safe, effective, and affordable ZIKV vaccine remains a global health priority (Shan et al., 2018). Several ZIKV vaccine strategies have been pursued, including live-attenuated virus (Kwek et al., 2018; Shan et al., 2017; Xie et al., 2018) subunit (Medina et al., 2018; Tai et al., 2019), inactivated (Larocca et al., 2016; Modjarrad et al., 2018), DNA (Abbink et al., 2016; Dowd et al., 2016; Tebas et al., 2017), mRNA (Pardi et al., 2017; Richner et al., 2017)), and viral vectors Adenovirus (Bullard et al., 2020; Larocca et al., 2019), Measles (Nurnberger et al., 2019), and VSV (Emanuel et al., 2018)), but to date no licensed vaccine is available. Historically, live-attenuated viral vaccines induce long-lasting protective immunity but have reduced safety, whereas inactivated vaccines provide a high level of initial safety but exhibit weak long-term immunity (Amanna and Slifka, 2009; Lambert et al., 2005). To overcome the trade-off between a sustained immunity and vaccine safety, the concept of a vaccine based on single-cycle, encapsidation-defective flaviviruses was developed (Pang et al., 2014; Widman et al., 2009). These third-generation flavivirus vaccines were surprisingly potent, with a high level of safety in animal models(Widman et al., 2008). However, the generation of the single-cycle, encapsidation-defective viruses involved the deletion of a large portion of the capsid (C) protein, requiring either a complicated packaging system (Pang et al., 2014) or helper viral replicon (Mason et al., 2006) for replication. Such packaging systems make commercial vaccine production difficult and costly. In this study, we sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus to overcome the dependence upon packaging cells for production of single-cycle viral vaccine.

Insect-specific viruses are thought to be ancestral to arboviruses (Öhlund et al., 2019). However, the detailed mechanism(s) of insect-specific flavivirus (ISF) host-range restriction remains poorly understood. The vertebrate host restriction of ISF may occur at the level of viral attachment and entry, genome replication, viral assembly, or release (Junglen et al., 2017; Piyasena et al., 2017). Our goal is to generate a highly immunogenic ZIKV mutant for vaccine development. That virus should be capable of expressing most or all ZIKV antigens without producing infectious viral progeny in a vertebrate host. For ZIKV and related flaviviruses, the final cleavage of the glycoprotein prM to generate its mature form M is performed by host protease furin (Pierson and Diamond, 2012; Roby et al., 2015; Yu et al., 2008; Zybert et al., 2008b). Furin is a cellular endoprotease that proteolytically activates large numbers of proprotein substrates, primarily in secretory pathway compartments. We noted that dual-tropic flaviviruses exhibit low cleavage efficiency of prM and produce a high proportion of immature and partially mature viral particles. By way of example, for dengue virus, the immature and partially mature viruses induce a high level of antibodies against prM, which in turn enhances subsequent dengue virus infection of a different serotype (Rodenhuis-Zybert et al., 2011). For ZIKV and other dual-tropic flaviviruses, the evolutionary advantage for the suboptimal cleavage of prM is not clear. By comparison, ISFs are very divers in furin cleavage site in prM. Some of ISFs contain highly optimized furin cleavage sequence in prM. We hypothesize that ZIKV may have adapted to replicate efficiently in vertebrate cells by maintaining a suboptimal cleavage site in prM, and consequently the optimization of the cleavage site in prM may preferentially reduce virus fitness in vertebrate cells. To verify this hypothesis, reverse genetics was used to introduce specific mutations into the furin cleavage sequence within the RNA of the ZIKV prM. Specifically, the furin cleavage motif of the ZIKV prM was gradually modified to investigate whether mutations in the cleavage site could influence the viral replication in vertebrate cells and insect cells.

2. Materials and Methods

2.1. Cell cultures, virus stocks, E. coli, yeast strains, and antibodies.

Vero cells (African Green Monkey Kidney Epithelial Cells) and Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured as previously described (Markoff et al., 2002; Pang et al., 2001b). ZIKV (MR766 strain) was purchased from ATCC. Frozen, competent E. coli strain Stbl4™ was purchased from Thermo Fisher Scientific (Waltham, MA). Saccharomyces cerevisiae YPH857 (ATCC® 76628™) and murine monoclonal antibody (mAb) 4G2 (ATCC® VR-1852™) were acquired from the ATCC, A murine mAb to ZIKV NS5 was raised in our laboratory. Goat anti-mouse IgG conjugated with Alexa Fluor 488 was obtained from Millipore Sigma (St Louis, MO).

2.2. Generation of replication restricted mutant ZIKVs by reverse genetics.

We generated an infectious full-length cDNA clone of ZIKV MR766 based on a modified pRS424 (ATCC® 77105™) yeast shuttle vector (supplementary data). To facilitate the construction of mutant ZIKVs, a unique Not I site was created in the full-length cDNA clone. At the same time, the ZIKV sequence from 200bp to 1200bp was removed. The modified clone was used as a basis for constructing mutant ZIKVs since Not1 is a “rare cutter” that provided a unique site for linearizing the plasmid. DNA fragments bearing the designed mutation were created by PCR. The mutations were introduced into the ZIKV MR766 cDNA clone by homologous recombination between a PCR fragment bearing the mutations and linearized ZIKV plasmid in yeast cells as described previously (Markoff et al., 2002). The oligomers used and an overview of the process are shown in figure 1 and in the Supplementary Data. The mutant ZIKV plasmids were verified by DNA sequencing. In vitro transcription of mutant ZIKV was catalyzed by SP6 RNA polymerase (Promega Corporation, Madison, WI.) in the presence of a cap analog, using linearized mutant ZIKV cDNA clone as templates. C6/36 cells were transfected with TransIT®-mRNA Transfection Kit (Mirus Bio, Madison, WI) according to the manufacturer’s instructions. Mutant viruses were harvested from the transfected insect cells 7 days post-transfection.

FIG. 1. Generation of mutant cDNA clones.

FIG. 1.

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A unique Not I site was created in an infectious full-length cDNA clone. At the same time, the ZIKV sequence from 200bp to 1200bp was removed. The mutations were introduced into the ZIKV MR766 cDNA clone by homologous recombination between a linear PCR fragment bearing the mutations and linearized ZIKV MR766 plasmid in yeast cells

2.3. Isolation of a stable vertebrate-specific replication-defective ZIKV (VSRD-ZIKV) by selective adaptation

C6/36 cells were seeded in 96-well plates (Corning, US) and incubated overnight at 28°C. Virus from ZIKV-MS2 stock was diluted with medium to a concentration of 2 × 104 FFU/ml. Serial 1:2 dilutions of the virus were made in the first column (rows A1-H1), followed by 1:2 dilutions horizontally (columns 1–12). The volume of each well was adjusted to 200uL. The plates were incubated at 28°C. After five days, 5 μl of the virus from each well of the 96-well plate was used to infect Vero cell monolayers in 8-well Lab-Tek chamber slides (Thermo Fisher Scientific, Waltham, MA). The wells on the original 96 well plate that contained the VSRD-ZIKVs were identified by immunofluorescent assay of Vero cell in the 8-well chamber slides. The VSRD-ZIKVs were selected and passaged on C6/36 cells in a T-25 flask for adaptation. We repeated the isolation and adaptation process until a stable vertebrate-specific replication-defective-ZIKV was isolated (VSRD-ZIKV).

2.4. Indirect Immunofluorescence Assays.

Indirect immunofluorescence assay (IFA) was performed to detect viral protein expression in Vero cells and C6/36 cells. Cells transfected with viral RNA or infected by viruses were grown on 8-well chamber slides. At the indicated time points, the cells were fixed in acetone at −20°C for 15 min. The cells were treated with a 4G2 flavivirus group-specific mAb or NS5 mAb antibody for 1 h and washed three times with PBS (5 min for each wash). Cells were then incubated with Alexa Fluor 488™ goat anti-mouse IgG for 1 h in PBS buffer and washed three times with PBS. The processed 8-chamber slides were observed and photographed with a fluorescent microscope.

2.5. Viral Titration by Plaque Assay.

Vero or C6/36 cells monolayer in 12-well plates were infected with serial 10-fold dilutions of virus for one h at 37°C (for Vero cells) or 28°C (for C6/36 cells). The cells were washed with serum-free DMEM and cultured for 3 to 5 days in DMEM containing 3% fetal bovine serum and 2% sodium carboxymethyl cellulose (Sigma). After 3–5 days, cell monolayers were fixed with 4% formaldehyde and incubated at room temperature for 2h. The fixative was removed, and the plate was stained with 1% crystal violet for 1 hour. Visible plaques were counted, and the viral titers were calculated. The titer of viruses used for infection in this study was based upon plaque assay in C6/36 cells. All data are expressed as the means of triplicate samples.

2.6. Fluorescent focus assay and immunostaining.

The fluorescent focus assay was performed by serial tenfold dilutions (101–106) of viral samples in DMEM. For each dilution, 100 μl of the sample was added to an 8-well chamber slide containing about 90% confluent cells. After a one hour incubation at 37°C (for Vero cells) or 28°C (for C6/36 cells), the fluid was removed, and 300 μl of methylcellulose containing 2% FBS and 1% penicillin-streptomycin was overlaid on the cells. The chamber slides were incubated for 48 h, after which the methylcellulose overlay was removed. The cells were fixed in acetone at −20°C for 15 min. After removing the fixative solution, the chamber slides were air-dried, washed three times with PBS, incubated in PBS with 1% FBS for 1 h, and reacted with a murine monoclonal antibody either 4G2 or NS5 mAb for 1 h. The chamber slides were washed three times with PBS, and then the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG for 1 h in PBS. The antibody was removed, and the cells were washed three times with PBS. Samples were observed with a Leitz fluorescent microscope, and the positive cell number calculated. The titer of viruses used in the animal study was based IFA in C6/36 cell s. All data are expressed as the means of triplicate samples.

2.7. In vivo evaluation of VSRD-ZIKV in immunodeficient AG129 mice.

All animal experiments were approved by the Huazhong Agricultural University IACUC. AG129 mice were obtained under specific pathogen-free conditions from the Wuhan Institute of Virology. 3-week-old AG129 mice (N=5/group) were infected with 10 FFU, 1 × 102 FFU of the ZIKV-MR766 wildtype strain, or either 1 × 105 FFU, or 1 × 106 FFU VSRD-ZIKV via the intraperitoneal injection. DMEM was injected via the same route as a mock-infection control. Disease progression in mice was monitored by weight loss and death. To measure viremia, mice were anesthetized and bled through the caudal vein every other day. The potential for neurovirulence was evaluated in newborn AG129 mice. Three groups of 1-day old AG129 mice (n = 12, ea.) were injected intracranially with indicated amounts of DMEM, VSRD -ZIKV, or ZIKV MR 766. Mice were monitored daily for morbidity and mortality for a period of 30 days.

2.8. Quantitative real-time PCR assays.

Total RNA was extracted from cells or cell supernatants by using Trizol reagent (Invitrogen, CA) The cDNA was synthesized by reverse transcription using the ReverTra Ace RT kit (Toyobo USA, NY). qRT-PCR was performed using the QuantStudio 6 Flex PCR system (Applied Biosystems, CA) and SYBR green PCR master mix (Toyobo). Cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 55°C for 10 s, and 65°C for 45 s. 10-fold serial dilutions of the cDNA of ZIKV MR766 wt (the titer have calculated by the plaque assay) worked as the standard were included with each qRT-PCR assay. The viral RNA concentration was determined by interpolation onto the curve made up of 10-fold serial dilutions of the standards.

2.9. 1. Statistical analysis.

All experiments were carried out a minimum of three times under similar conditions. Analyses were conducted using GraphPad Prism, version 7 (GraphPad Software, CA). Statistical differences among the experimental groups were determined using the two-way analysis of variance (ANOVA) with subsequent Student’s test. A P-value of < 0.05 was considered significant.

2.9.2. Data availability

All data generated or analyzed during this study are included in this published article.

3. Results

3.1. Generation of Infectious cDNA Clones of Wildtype and Mutant ZIKVs

In this study, we used the prototype ZIKV MR766 strain to construct cDNA fragments representing the entire genome. RT-PCR primers were designed to allow amplification of ZIKV genomic RNA into five cDNA fragments of 2 to 3 kb with unique restriction sites located at both ends of each cDNA (Supplementary Data). The five cDNA fragments were first cloned into two plasmids with classic method (supplementary data). The cloned cDNA fragments were verified by DNA sequencing. The full-length ZIKV MR766 cDNA clone was assembled through homologous recombination. Replication of the virus after transfection of Vero cells with RNA derived from the cDNA clone indicated that we had generated an infectious full-length cDNA clone of ZIKV MR766 (Figure S1). Genome-wide DNA sequencing of the full-length cDNA of plasmid-derived ZIKV confirmed that no mutations had been introduced into the full-length clone during passage in Vero cells.

To facilitate the introduction of mutations into the viral prM region, a unique Not I site was inserted into the infectious full-length cDNA clone of ZIKV MR766 at position replacing ZIKV sequence from 200bp to 1200bp (Figure S3). DNA fragments bearing the designed mutation were created by PCR. The mutations were introduced into the ZIKV MR766 cDNA clone by homologous recombination between PCR fragment bearing the mutations and linearized ZIKV plasmid in yeast cells as described previously (Markoff et al., 2002; Pang et al., 2001a)(Figure 1). The mutant ZIKV plasmids were verified by DNA sequencing.

Structural and functional mapping of the cleavage preferences of furin revealed the importance of both short-range (P4-P1) and long-range (P14-P5 and p1′-p4′) interactions in defining furin cleavage (Izidoro et al., 2009; Shiryaev et al., 2013; Tian et al., 2012). Therefore, we constructed mutants with different amino acids R and H in the region of the furin cleavage site. The ZIKV-H and ZIKV-R viruses are examples of that approach (Table S4). Additionally, a series of chimeric ZIKVs with ISF sequence in the furin cleavage site was developed to determine whether mutants could affect the production of infectious virus in vertebrate cells. Like ZIKV, cells infected with Dengue virus (DENV) secrete high levels (~30%) of prM-containing immature particles (Zybert et al., 2008a) suggesting that cleavage of prM is not efficient. Our previous work demonstrated that R mutation at P3 position of furin cleavage site in prM results in a mutant DENV serotype 2 (DENV2) completely replication-defected in vertebrate cells. Considering the close relationship between DENV and ZIKV, we, therefore, constructed ZIKV mutants from the furin cleavage region of an ISV DENV2 was substituted for the furin site in ZIKV. The representative mutant ZIKV constructs were shown in supplementary table S4.

3.2. Development of stable Vertebrate-Specific Replication-Defective ZIKV by selective adaptation

To investigate how ZIKV replication was influenced by the substrate specificity of furin enzymes in both arthropod and vertebrate cells, mutant ZIKV viruses were generated from above cDNA constructs. Mutant ZIKV genomic RNAs were synthesized by in vitro transcription as described previously (Markoff et al., 2002). Since we were looking for mutant viruses with vertebrate-specific growth restriction for vaccine development, mosquito-derived C6/36 cells were transfected with the mutant viral RNA derived from the cDNA constructs. Mutant ZIKVs were collected seven days post-transfection. When compared to the wildtype parent virus with the mutant ZIKVs contained point mutations, the mutant virus ZIKV-R, ZIKV-H, and ZIKV-6R exhibited little effect on viral growth in C6/36 cells but had reduced replication kinetics and peak titers in Vero cells (Table S4). In addition, ZIKV-HR further reduced the virus fitness in Vero cells specifically (Figure 2). These results suggested that H at P7, R at P6, and R at P3 of furin cleavage site in ZIKV prM were important for the restricted replication of ZIKV in Vero cells, and the effects were additive or synergistic in nature. Interestingly, the mutant virus ZIKV-M1 and ZIKV-M2 contained large piece of ISV DENV2 sequences showed the most reduced virus growth in Vero cells but with wildtype-like replication efficiency in C6/36 cells (Figure 2 and Table S4). However, it is currently unknown how the mutant amino acids (AA) outside furin cleavage site influenced prM cleavage and ZIKV fitness in Vero cells.

FIG. 2. Growth kinetics of the parent ZIKV and selected mutant viruses in C6/36 or Vero cells.

FIG. 2

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(A) A diagram of the ZIKV genome indicates the location of the genes for the structural (Yellow) and non-structural proteins (Green). The polypeptide sequences for the region of prM of ZIKV MR766 and mutant viruses. (B) ZIKV MR766 and mutant isolates were used to infect both C6/36 (mosquito) and Vero (non-human primate) cells at an MOI of 0.1. Virus titer was measured daily over 5- or 6-days post-infection, as described in the Methods section.

Next, virus stocks from the mutant ZIKVs were analyzed by IFA. Surprisingly, the immunofluorescence images of ZIKV-HR, ZIKV-M1, and ZIKV-M2 infected Vero cells showed significant more single positive fluorescent cells but less fluorescent foci than wildtype ZIKV infected Vero cells (Figure S4). The multicellular fluorescent foci were formed when virus-infected cells were able to produce infectious progeny virus, and the progeny virus, in turn, infects the surrounding cells. The single positive fluorescent cells are cells infected by a virus, which cannot produce infectious progeny virus. Therefore, the ZIKV-HR, ZIKV-M1, and ZIKV-M2 virus stock might contain viruses with both replication-capable and replication-defective viruses when test in Vero cells. Since all the three mutant viruses and wildtype ZIKV showed similar fluorescent image patterns in infected C6/36 cells, we concluded that “ZIKV-HR, ZIKV-M1, and ZIKV-M2 virus stock contained a mixture of dual tropism and single tropism viruses. Comparing the number of fluorescent foci to single positive cells relative, ZIKV-M2 produced the lowest percentage of infectious viruses in Vero cells. To obtain a stable vertebrate-specific replication-defective ZIKV, we adapted the ZIKV-M2 mutant to insect cell replication but no growth in vertebrate cells by multiple cycles of replication in C6/36 as described in the Methods section. After adaptation, we obtained one clone of ZIKV that replicated efficiently in C6/36 insect cells but did not produce infectious virions in Vero cells. We further evaluated the growth of that isolate in human (HeLa, U251) and baby hamster kidney (BHK-21) cell lines. The titer of wildtype ZIKV MR766 was approximately 105 pfu/mL, while no recoverable virus was released by VSRD-ZIKV (Figure 3 A). The fluorescent-positive cells observed in the tested cell lines indicated that this mutant ZIKV did not appear capable of spreading in vertebrate cells (Figure 3 B). Additional experiments using qRT PCR to measure intra and extracellular viral RNA in Vero and C6/36 cells showed that Vero cells infected with VSRD-ZIKV had intracellular levels of viral RNA comparable to ZIKV-MR-766. (Figure 4 B). In contrast no VSRD-ZIKV viral RNA was detectable in the mutant infected Vero cells supernatant (Figure 4 D).

FIG. 3. Characterization of VSRD-ZIKV and wildtype ZIKV replication in vertebrate cells.

FIG. 3.

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(A) The complete absence of infectious virus demonstrated that VSRD-ZIKV was replication-defective in (i) Baby hamster kidney (BHK-21) cells, (ii) Human cervical tumor (HeLa) cells, (iii) Human glioblastoma tumor (U251) cells. (B) Immunofluorescent staining of parallel infected cell cultures 36 hours p.i. indicated that VSRD-ZIKV might initiate only a single round of infection in the vertebrate cells tested.

Fig. 4. Quantitative real-time PCR assays of infected cells and cell-free medium from infected cells.

Fig. 4.

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ZIKV MR766 and VSRD-ZIKV were used to infect both C6/36 and Vero cells at an MOI of 0.1. Virus titer of infected cells (A and B) and supernatant (C and D) was measured at indicated time points using quantitative real-time PCR. Means and SDs from three independent replicates are shown. Statistics were performed using unpaired Student’s t test; *significant (p < 0.05); **highly significant (p < 0.01).

3.3. DNA sequencing of VSRD-ZIKV and analysis of new engineered mutations

DNA sequencing analysis of the VSRD-ZIKV genome revealed that the adaptation process introduced two additional amino-acid (AA) mutations into ZIKV-M2. Interestingly, both mutations were in the multi-functional viral non-structural protein 3 helicase (NS3) region (Fig. 5 A). The stability of VSRD-ZIKV was confirmed by 10 serial passages in C6/36 cells, with the result that no additional mutation was observed by genome-wide cDNA sequencing. Thus, we generated a stable VSRD-ZIKV through a combination of reverse genetics and selective adaptation.

Fig. 5. Characterization of the ZIKV-M2-derived mutant viruses.

Fig. 5.

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(A) Diagram of the viral genome and polypeptide sequences for ZIKV MR766 and ZIKVs derived from mutant clone ZIKV-M2. (B) Growth kinetics of the parental ZIKV MR766 and mutant viruses in C6/36 cells or Vero cells indicated that both prM and NS3 mutations were required for the stable inhibition of VSRD-ZIKV in Vero cells.

We used reverse genetics to evaluate the role of NS3 mutations in enhancing VSRD-ZIKV stability in C6/36 cells, using three mutant viruses derived from the ZIKV-M2 construct (Figure 5 A). IFA analysis of virus stocks from the mutant viruses ZIKV-M2–291N and ZIKV-M2–452A showed some fluorescent foci in infected Vero cells, in addition to large number of single positive cells. In contrast, the IFA image of ZIKV-M2–291N-452A infected Vero cells shown only single positive cells (data not shown). We monitored the growth kinetics of the mutant viruses ((ZIKV-M2–291N, ZIKV-M2–452A, and ZIKV-M2–291N-452A) in C6/36 and Vero cells. The replication of mutants with either the 291N or 452A mutations was well below that of ZIKV-MR766. In contrast, the ZIKV-M2–291N-452A mutant did not replicate in Vero cells. This approach supports the hypothesis that both mutations were required for maintaining the VSRD-ZIKV stability in C6/36 cells (Figure 5 B)

3.4. Preliminary evaluation of VSRD-ZIKV in both newborn and 3-week-old immunocompromised mice

To assess the in vivo safety of the mutant VSRD-ZIKV, we used interferon α/β and γ receptor-deficient AG129 mice. These mice are highly susceptible to WT ZIKV infection and succumb to infection with a dose as low as 1 FFU(Aliota et al., 2016). Two groups of five three-week-old AG129 mice received either 105 or 106 FFU of VSRD-ZIKV via the intraperitoneal route (ip). The two control groups were injected with 10 or 100 FFU ZIKV MR766 with same route. The ZIKV MR766-infected group developed detectible viremia and died eight days post-infection. In contrast, all the VSRD-ZIKV-infected mice survived and had no detectible viremia (Fig. 6A–C). VSRD-ZIKV mice did not exhibit sickness or weight loss throughout the study (30 days). We then evaluated the potential neurovirulence of VSRD-ZIKV in newborn AG129 mice following an intracranial injection (ic). Two groups of one-day-old AG129 mice (n = 12 per group) were injected with 104 or 105 FFU of VSRD-ZIKV via ic. Mice in the control group were infected with 10 FFU ZIKV MR766 via same route. Mice in the sham infected group were injected with 20μL DMEM via ic. The VSRD-ZIKV-infected mice and sham group mice survived, developed normally, and did not exhibit neurological defects. In contrast, the mice in the ZIKV MR 766 group died within five days (Figure 6 D).

FIG. 6. VSRD-ZIKV infection of AG129 mice.

FIG. 6.

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Three-week-old AG129 mice (N = 5 per group) were infected with the parent ZIKV MR766 (10 or 100 FFU), VSRD-ZIKV (105 or 106 FFU), or sham by the subcutaneous route. Following viral infection, body weight (A) and survival (B) were monitored. The serum viral loads in mice that received VSRD-ZIKV and 100 FFU ZIKV MR766 were determined. (C) One-day-old AG129 mice (N = 12 per group) were sham-infected or challenged with parent ZIKV MR766, or VSRD-ZIKV by the intracranial route. The survival, of AG129 mice infected with wildtype and VSRD-ZIKV is shown in panel D.

4. Discussion

Arboviruses exhibit dual host tropism and are transmitted from insects to their vertebrate hosts during the arthropod host’s blood-feeding (Olmo et al., 2019). The majority of known arboviruses belong to viral families such as Flaviviridae, Togaviridae, and Phenuiviridae. Moreover, each family has members that are insect-specific or monospecific viruses, which are thought to be ancestral arboviruses(Cook and Holmes, 2006; Cook et al., 2012; Marklewitz et al., 2015). Due to the increasing threat of emergence and re-emergence of arboviruses capable of infecting humans, we and others (Calisher and Higgs, 2018; Halbach et al., 2017) felt it is vital to investigate the evolution of the switch from a single host to dual-host. Previous studies have shown that the host range restriction of ISVs occurs at several levels of the viral life cycle (Junglen et al., 2017). By way of example, a recent report found that a ZIKV with a mutant envelope protein 316Q/461G had reduced growth in vertebrate cells (Setoh et al., 2019). Additionally, the 316Q/461G mutant grew at the same rate as wildtype ZIKV when Vero cells were incubated at 28 degrees C, the temperature that insect cells are normally grown at, suggesting the change may be temperature sensitive. Furthermore, the envelope mutant was not replication defective but growth restricted in Vero cells (Setoh et al., 2019). In this study, we described a novel reverse genetics-based approach to generate a vertebrate cell-specific replication-defective ZIKV by targeting the furin cleavage site within the viral prM glycoprotein. Furin is a cellular endoprotease that proteolytically cleaves a large number of proprotein substrates, including the prM glycoprotein. The furin-mediated cleavage of prM is a critical step for flavivirus maturation. However, vector-borne flaviviruses exhibit low efficiency cleavage of prM and produce a high proportion of immature and partially mature viral particles. The evolutionary advantage for the dual-host flaviviruses to adapt to suboptimal furin cleavge site in prM is not clear. Here, we provide evidence that optimized fuin cleavage site in prM preferentially reduced ZIKV fitness in vertebrate cells.

Our initial series of mutations surrounding the furin cleavage site in prM which may enhance processing of prM resulted in reduced replication in Vero cells. However, upon further investigation, we determined that the virus produced from insect cells was a mixture of viruses that replicated either normally or were replication defected in Vero cells. We then used adaptation and selection for virus isolates, which were unable to replicate in Vero cells. Like natural ISVs, one virus clone, VSRD-ZIKV, was capable of replication and production of infectious virus in insect cells but not in any of the vertebrate cells tested (Figure. 3). The strict insect cell tropism of VSRD-ZIKV was maintained through multiple (10) cycles of replication in C6/36 insect cells. Whole genome sequencing of the VSRD-ZIKV mutant cDNA indicated that in addition to the engineered mutations in the furin cleavage site of the ZIKV MS2 parental virus: mutations occurred at positions 291 and 452 within the non-structural NS3 protein. Using our reverse genetics platform, we engineered three mutant viruses (ZIKV-M2–291N, ZIKV-M2–452A, and ZIKV-M2–291N-452A) that were derived from the ZIKV-M2 construct. Only the ZIKV-M2–291N-452A was completely and stably replication defected. This observation indicates that while the cellular furin proteinase cleavage site played a role in the VSRD-ZIKV tropism change, it may not be the only mechanism in play.

The viral assembly and maturation pathway for flaviviruses is not clearly understood. However, we have appeared to have orchestrated changes in the sequences of the ZIKV surface glycoproteins that may affect the proteolytic cleavage of prM protein by host protease furin. Our data suggest that furin may play a significant role in the pathway to produce infectious viruses. The engineered mutations in the sequence of the furin cleavage site in the prM appeared to limit the virus release in vertebrate, but not C6/36 insect cells (Figure 2,). Further analysis of ZIKV RNA by qRT-PCR of infected cells and cell-free supernatant fluid in VSRD-ZIKV and wildtype virus infected cells indicated that VSRD-ZIKV could replicate its RNA intracellularly (Figure 3). However, no viral RNA was detectible in the supernatant fluid from VSRD-ZIKV infected Vero cells. These qRT-PCR data, combined with indirect immunofluorescence assay detection of viral protein expression in VSRD-infected Vero cells suggest that VSRD-ZIKV may only be capable of a single round of infection in vertebrate cells. While further studies are required to elucidate mutational changes in viral maturation in Vero cells, preliminary electron microscopy images have suggested that the enhanced furin cleavage efficiency of prM prevent immature virus particle formation, but forming virus like particles in vertebrate cells infected with VSRD-ZIKV (Data not shown). It is not clear how these mutations in the NS3 protein of the VSRD-ZIKV and ZIKV-M2–291N-452A effect virus stability in C6/36 cells. Importantly, this observation may lead to a better understanding of the viral maturation process as well as changes related to the switch from an ISV to a vertebrate tropic virus.

To investigate the replication of VSRD-ZIKV in vivo, we selected AG129 mice. Immunodeficient AG129 mice are a highly sensitive lethal model because these mice succumb to infection with a dose as low as 1 IFU wildtype ZIKV (Aliota et al., 2016). The high dose of VSRD-ZIKV (106 FFU) used to challenge of both newborn, and 3-week-old immunocompromised mice demonstrated that the VSRD-ZIKV was neither lethal nor neurovirulent. It appears that VSRD-ZIKV can only initiate single-round infection and express all viral antigens in vivo, similar to a live virus vaccine. On the other hand, infection with VSRD-ZIKV grown in insect cells would not produce infectious virus in the vertebrate host, which may yield a safety level similar to inactivated vaccines.

Vaccine development for vector-borne infectious viruses is a top public health priority due to the significant global disease burden (Christou, 2011; Martina et al., 2017). While no human vaccine has been licensed for ZIKV, there are effective live, attenuated vaccines available for the related mosquito-borne viruses YFV (Beck and Barrett, 2015), DENV (Guirakhoo et al., 2004), and JEV (Chen et al., 2015; Chokephaibulkit et al., 2010). Inactivated vaccines are also licensed for JEV (Jelinek, 2009) and the tick-borne flaviviruses tick-borne encephalitis (TBEV) (2011) and Kyasanur forest disease (KFDV) (Dandawate et al., 1994). In response to the explosive ZIKV epidemic in the Americas, several vaccine platforms have been investigated, including inactivated virus (Larocca et al., 2016; Modjarrad et al., 2018), live attenuated virus (Xie et al., 2018), DNA vaccine (Abbink et al., 2016; Tebas et al., 2017) and RNA vaccines (Pardi et al., 2017; Richner et al., 2017).

From a vaccine development perspective, VSRD-ZIKV can be grown to high titer in insect cell lines, which will enable large-scale and cost-effective vaccine production. Preliminary studies with VSRD-ZIKV suggest that this ISV can elicit potent cellular and humoral immune response in our murine model and protected mice against lethal challenge of both ZIKV MR766 and ZIKV of Puerto Rico (PRVABC59) strain (Wan et al., in preparation). Future research will determine if long-term immunity can be induced with VSRD-ZIKV. Furthermore, the methods described in the present study can be potentially applied to other vector-borne viruses for the development of safe, effective, and affordable vaccines.

Supplementary Material

Supplementary

Acknowledgments

We are grateful to Drs. Bruce Jones, Andrea Jackson, and Esther Childers for their remarkable support and guidance. We acknowledge Mrs. Dan Zhang for technical support. We thank Science Docs Inc. for language editing. This project was in part funded by National Institutes of Health Grants P20CA192989 and R41AI129119

Footnotes

Ethics declarations

Competing interests

The authors declare no competing interests.

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